MINI REVIEWpublished: 22 March 2019

doi: 10.3389/fendo.2019.00167

Frontiers in Endocrinology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 167

Edited by:

Angel Nadal,

Universidad Miguel Hernández de

Elche, Spain

Reviewed by:

Isabel Navarro,

University of Barcelona, Spain

Paloma Alonso-Magdalena,

Universidad Miguel Hernández de

Elche, Spain

*Correspondence:

Bruce Blumberg

blumberg@uci.edu

Specialty section:

This article was submitted to

Systems and Translational

Endocrinology,

a section of the journal

Frontiers in Endocrinology

Received: 21 November 2018

Accepted: 28 February 2019

Published: 22 March 2019

Citation:

Chamorro-Garcia R and Blumberg B

(2019) Current Research Approaches

and Challenges in the Obesogen

Field. Front. Endocrinol. 10:167.

doi: 10.3389/fendo.2019.00167

Current Research Approaches andChallenges in the Obesogen Field

Raquel Chamorro-Garcia 1 and Bruce Blumberg 1,2,3*

1Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, United States, 2Department of

Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, United States, 3Department of Biomedical Engineering,

University of California, Irvine, Irvine, CA, United States

Obesity is a worldwide pandemic that also contributes to the increased incidence of

other diseases such as type 2 diabetes. Increased obesity is generally ascribed to positive

energy balance. However, recent findings suggest that exposure to endocrine-disrupting

chemicals such as obesogens during critical windows of development, may play an

important role in the current obesity trends. Several experimental approaches, from in

vitro cell cultures to transgenerational in vivo studies, are used to better understand

the mechanisms of action of obesogens, each of which contributes to answer different

questions. In this review, we discuss current knowledge in the obesogen field and the

existing tools developed in research laboratories using tributyltin as a model obesogen.

By understanding the advantages and limitations of each of these tools, we will better

focus and design experimental approaches that will help expanding the obesogen field

with the objective of finding potential therapeutic targets in human populations.

Keywords: obesogens, obesity, MSCs, 3T3-L1, transgeneration, isoDMBs, tributyltin, metabolism

In the last 40 years obesity rates have dramatically increased worldwide both in adults and in youth(1). A recent report on obesity trends in the U.S. estimated that 39.8% of adults are clinically obese(BMI > 30) (1). Importantly, obesity is a risk factor for other metabolic disorders such as type 2diabetes whose prevalence has increased in parallel with obesity and which is predicted to affect 642million people worldwide by 2040 (2, 3). The health costs associated with obesity were estimatedat over $275 annually in the U.S alone (4). Although the major driving factor in obesity is usuallyconsidered to be a simple function of energy balance (calorie consumption higher than calorieexpenditure) (5), recent reports showing the increasing trends of obesity in children under 1 year ofage and in animal populations suggest that other factors may be playing important roles in obesity(6, 7). Understanding those factors, the windows of susceptibility and the mechanisms throughwhich they to alter human metabolism and promote obesity will aid treating and preventing theincreasing rates of obesity and related disorders worldwide.

Obesity is sexually dimorphic (8). Overall, females accumulate more fat than males, and ittends to be located subcutaneously, while in males, adipose tissue tends to accumulate in thevisceral cavity. Subcutaneous adipose tissue is generally associated with healthier fat since it isinvolved in regulating thermogenesis while visceral adipose tissue is associated with increasedrisk of cardiovascular disease (9). Adipose tissue is now known not only for its ability to storelipids, but also for its contribution to metabolic homeostasis by secreting hormones (e.g., leptinand adiponectin) and other signaling molecules involved in the regulation of appetite and fatmobilization. This suggests that maintaining a healthy and functional adipose tissue improves theoverall metabolic state of the individual (9). The sexually dimorphic content and distribution offat is highly influenced by steroid hormones such as testosterone and estrogens (8). Therefore,

Chamorro-Garcia and Blumberg Challenges in the Obesogen Field

alterations of the endocrine system may contribute todisturbances in the regulation of adipose tissue formationand maintenance.

Endocrine disrupting chemicals (EDCs) are exogenouschemicals that alter the natural function of hormones (10).Several human and animal studies have found associationsbetween exposure to EDCs and increased adiposity (11–16).However, the effects of EDCs that may lead to obesity mightnot be linked exclusively to life style or environmental exposuresduring adulthood. Epidemiological studies have shown thatthe environment during in utero development may affect theprevalence of non-communicable diseases, including metabolicdisorders, later in life (13–21). Notably, the nutritional state of themother is strongly linked to body weight of the offspring at birthand later in life (22–26) suggesting that suboptimal environmentsduring development may contribute to permanent alterationsin the individual that will counteract any lifestyle actions toameliorate weight gain, thereby increasing susceptibility toobesity (22, 27).

A large body of evidence shows that exposure toenvironmental pollutants during in utero development andearly life may contribute to obesity later in life (11). Studiesperformed using both in vitro and in vivo models showed thata subset of endocrine disrupting chemicals, called obesogens,can have important effects on the development of adiposetissue. Obesogens are exogenous chemicals that inappropriatelystimulate adipogenesis and fat storage, can disturb adipose tissuehomeostasis and affect metabolic rates and/or the regulationof appetite and satiety (28). Our recent findings suggestedthat one consequence of these disturbances is the alteration ofthe metabolic setpoint of the individual, that is, the capabilityof the body maintain a particular weight regardless of thelifestyle changes taken to increase or reduce that weight (29).These results are supported by human studies showing anassociation between higher levels of perfluoroalkyl compoundsin plasma and a more rapid weight regain after diet-inducedweight loss plan (12). Moreover, individuals with the highestlevels of perfluoroaklyl compounds has the lowest restingmetabolic rate (12). Despite numerous studies demonstratingthe existence and effects of obesogens, a longstanding debateabout the mechanisms through which obesogens exert theirobesogenic effects still persists. In this review, we discuss thecurrent experimental approaches to study the mechanisms offunction of obesogens from in vitro analysis to transgenerationalin vivo studies.

IN VITRO APPROACHES

The prototypical obesogen tributyltin (TBT) is known toactivate two nuclear receptors critical for adipogenesis. Theseare the peroxisome proliferator-activated receptor gamma(PPARγ)(30–33), which is considered to be the master regulatorof adipogenesis (34), and its heterodimeric partner the retinoidX receptor (RXR)(30), which we recently showed to be animportant regulator of adipocyte commitment (35). Adipogeniccommitment is the process through which multipotent

mesenchymal stem cells (MSC) lose their multipotency andbecome irreversibly destined to the adipocyte lineage, in partvia the expression of PPARγ2. Activation of PPARγ2 elicitsterminal differentiation into white adipose tissue (36). Otheralready characterized obesogens such as fludioxonil, quinoxyfen,dibutyltin or triphenyltin, also activate PPARγ and/or RXR(37, 38), suggesting that these nuclear receptors are commontargets for EDCs. However, nuclear receptors other thanPPARγ2 and RXR have been shown to play important rolesin adipogenesis, including the glucocorticoid receptor (GR)(39), estrogen receptor (ER) (40) and androgen receptor (AR)(41) and obesogens such as tolylfluanid, bisphenol A (BPA) ordichlorodiphenyltrichloroethane (DDT) have been shown toact through them (42–44). The fact that estrogen and androgenreceptors are also involved in regulation of adipogenesis andfat storage, may explain, at least in part, the sexually dimorphicdistribution of the fat in individuals from different genders.Therefore, assessing the capability of a candidate obesogen toinduce adipogenic commitment, its ability to promote finaladipocyte differentiation, and the mechanisms through whichthese processes occur requires the use of an array of assays thattests the various potential paths used by obesogens to promotefat storage.

One frequently used tool to screen for new obesogens is themurine pre-adipocyte cell line 3T3-L1 (45). This immortalizedcell line has some beneficial aspects that include the short lengthof the assay and the amenability of cell lines to high throughputscreening approaches. However, one key limitation of 3T3-L1cells is that, since they are already pre-adipocytes, they arenot useful for examining mechanisms, including screening forchemicals that may be involved in adipocyte commitment. Itwas recently suggested that the commercial source of the cellline (e.g., ATCC or Zenbio), the type of plates used, as wellas the number of passages the cells have experienced play acritical role in the process of cell differentiation (46). 3T3-L1 cells from different commercial sources showed differentexpression of nuclear receptors such as PPARγ, RXRβ, and ERβ,and variable capability to differentiate into adipocytes using well-established adipogenic positive controls such as rosiglitazone(PPARγ activator) or TBT (46). These findings highlight someof the limitations of using 3T3-L1 cells as an adipogenic modelwhen the goal is to hypothesize about in vivo outcomes afterexposure to obesogens. Screening of new obesogens using thismodel will depend critically on poorly controlled variables suchas the source and number of passages of the cells, bovinesera, inconsistencies in coatings on tissue culture plates andother culture conditions which can confound reproducibility ofthe results.

In contrast, assays performed with uncommitted cells suchas MSCs (47), allow analyses with a broader scope. We recentlydescribed for the first time the role of RXR in adipogeniccommitment using MSCs as a cell model, revealing that thisphase of adipogenesis can be targeted by obesogens (35). Othershave used murine cell lines such as C3H/10T1/2 which resembleMSCs in some properties (but contain a karyotype of 80chromosomes), or BMS2 to screen for new obesogens or todive into adipogenic mechanisms through which obesogens may

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Chamorro-Garcia and Blumberg Challenges in the Obesogen Field

act. Although both cell types are accepted adipogenic models(48, 49), they are both immortalized cell lines that do notfully recapitulate the behavior of uncommitted precursors invivo. Both are also murine cells which can somewhat limit theextrapolation of results to other species. Therefore, conclusionsand inferences made from the use of these cells should accountfor such limitations. One advantage of using MSCs is that theycan be isolated as primary cells from different individuals in avariety of species allowing a more extensive analysis. The use ofhuman MSCs can also allow researchers to assess the variabilitythat exists in human populations, which would contribute toa deeper understanding of the role obesogens may play in thecurrent obesity pandemic.

IN VIVO APPROACHES

TBT has been shown to act as an obesogen in multiple organismsincluding mice (30–32, 50–52) rats (53), goldfish (54), andzebrafish (55) which supports the hypothesis that obesogens arelargely not species specific. However, although there are severalin vitro models available to study the molecular underpinningsof obesogen exposure, the mechanisms through which obesogenscontribute to obesity in animal models are likely to be morecomplex than those described for cell cultures. Experimentsperformed in adult rodents exposed to TBT showed alterationsof the hypothalamic-pituitary-gonadal axis (56) as well as non-alcoholic fatty liver (52) supporting the current knowledge thatthe regulation of metabolism requires coordination betweendifferent organs including fat tissue, liver, muscle, pancreas,or brain.

Another source of discussion in the obesogen field is relatedto the functionality of the adipocytes produced after exposureto obesogens (57). As mentioned above, the adipose tissue hasan important role to play in maintaining an overall healthymetabolic state (58). Interestingly, white fat cells producedby TBT exposure lack some beneficial properties of normalwhite adipocytes since the former show reduced levels of theantidiabetic hormone adiponectin, reduced glucose uptakecapability, impaired ability to develop into thermogenicbeige/brite adipocytes and increased expression of pro-inflammatory and pro-fibrotic genes (57, 59, 60). These resultssuggest that TBT promotes the development of dysfunctionaladipocytes (57, 59, 60). Understanding which obesogens produceadipocytes of normal function and which elicit the productionof dysfunctional adipocytes may also contribute to a betterunderstanding of the potential mechanistic differences betweendifferent obesogens.

TRANSGENERATIONAL APPROACHES

It has been shown that some obesogens including TBT,DDT, and BPA, are able to induce heritable changes thatare propagated through multiple generations without anyfurther exposure (29, 61–64). The non-Mendelian transmissionof these alterations suggests that epigenetic mechanisms areinvolved in this process. This raises yet another challenge

for the obesogen field: what are the mechanisms underlyingtransgenerational inheritance?

The field of environmental epigenetics studies theepigenetic alterations introduced by environmental factorsthat contribute to phenotypic variation and/or disease (65).Studies performed in animal models showed that exposureto obesogens during development increased predisposition toobesity not only in the directly exposed F1 and F2 generations(F2 are exposed as germ cells inside the F1 fetus) but alsoin subsequent F3 and F4 generations (and beyond) whichare not exposed (29, 61–64, 66, 67). These studies showedalterations of epigenetic factors such as DNA methylationand post-translational histone modifications in somatictissues and/or germ cells (29, 61–64, 66–68). However,there are key knowledge gaps in this transgenerationalpuzzle, including the characterization of the molecularmechanisms that occur in the directly exposed stages thattrigger the transgenerational phenotype, and the molecularmechanisms by which these alterations are transmitted throughmultiple generations.

There is extensive epigenetic reprogramming at differentstages of mammalian development. Shortly after fertilization,the first erasure of epigenetic marks (DNA methylation) occursfrom the zygote to the blastocyst during maternal-to-zygotictransition in a sex-specific manner (69). Between E 6.5 andE 13.5, after somatic and germ lines separate, the primordialgerm cells (PGCs) undergo another round of DNA methylationerasure while they travel to the genital ridge where they willmature into sex-specific germ cells (70). Considering that fromthe moment of fertilization until birth there is an extensiveepigenetic reprogramming of various epigenetic marks, thepresence of agents that may disturb these processes, such asobesogens and other EDCs, at any of the stages may contribute toalterations in the newly determined epigenetic landscape. Sincethis reprogramming occurs in every generation, it is critical todetermine which alterations are introduced by environmentalfactors and how many of these epigenomic changes canbe propagated to subsequent generations in order to betterunderstand mechanisms of transgenerational inheritance. This iscurrently a very controversial topic in the field (63, 65, 68, 71–73).

We recently proposed a new mechanism for the transmissionof epigenetic information. In our transgenerational paradigm, weexposed pregnant F0 female mice to environmentally relevantdoses of the obesogen TBT throughout pregnancy (61) orthroughout pregnancy and lactation (29). Following eitherapproach, we consistently found increased adiposity in F3 and F4males, but not females (29, 61). Interestingly, the differences infat storage between controls and ancestrally-TBT treated animalswas exacerbated when the diet of the animals was switched from astandard diet to a diet with higher fat content (29). This supportsthe hypothesis that ancestral exposure to TBT alters themetabolicset point of these animals multiple generations after exposure hasceased (29). Integrativemethylome and transcriptome analyses offat tissue isolated from obese F4 males did not reveal any directassociations between changes inmRNA expression levels of geneswhose promoter was either hypermethylated or hypomethylated.In contrast, we found a significant number of differentially

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methylated regions (DMR) located in intergenic regions. We re-analyzed the data, focusing on large blocks of the genome wherethe alteration in DNA methylation was in the same direction(hypo- or hyper-methylated) and denoted these as isodirectional,differentially methylated blocks (isoDMBs). IsoDMBs spannedlarge regions of the genome and we found a strong associationbetween hypomethylated isoDMBs and over-expressed genesand between hypermethylated isoDMBs and under-expressedgenes (29). Additionally, analysis of chromatin accessibility insperm samples from F3 and F4 mice showed significant overlapsin accessibility between sperm samples in both generations(Figure 1A), suggesting that there is a conservation of thedifferentially accessible regions across generations. We alsofound an intriguing association between chromatin accessibilityin sperm and isoDMBs in white adipose tissue of F4 malemice (Figure 1B). Regions that were inaccessible in sperm werehypomethylated in fat and regions that were accessible in spermwere hypermethylated in fat. We inferred that altered chromatinaccessibility in the germ cells may promote or permit differentialDNA methylation, epigenetic marks and gene expression insomatic tissues of the next generation (Figure 1C) (29).

We also noted that hypomethylated isoDMBs tend tobe located in areas of the genome with high GC content,whereas hypermethylated isoDMBs are located in areas of thegenome with low GC content (Figure 1D). In other words,rather than being located randomly distributed throughoutthe genome, changes in methylation were enriched in regionswith specific base composition (GC content) (29). Others haveshown that there is an association between base compositionand higher order chromatin organization in the nucleus (74).This led us to hypothesize that in utero exposure to TBT

causes heritable alterations in chromatin architecture that willcontribute to changes in the epigenetic landscape that arereflected in the transcriptome (29). This new model providesa potential molecular mechanism that embraces previouslydescribed results showing alterations of epigenetic marks, such asDNA methylation, histone modification and expression of smallnon-coding RNAs in germ cells and somatic cells after ancestralexposure to EDCs such obesogens (62–64, 66, 67).

WINDOWS OF SUSCEPTIBILITY

There are different windows of susceptibility to obesity afterobesogen exposure. Rodents exposed to obesogens such as TBTor nonylphenol during early adulthood showed increased ectopiclipid storage in liver elevated body weight, and altered levels ofmetabolic hormones (52, 75). BPA or tolylfluanid exposure ledto alterations in glucose metabolism and adiposity in adult mice(76, 77). Some of the metabolic alterations are only observed

when the animals are exposed to high fat diets or high fat/highsugar diets (75, 77), suggesting that obesogen exposure maybe increasing susceptibility to obesity in the presence of othermetabolic challenges. This has important implications for thehuman obesity pandemic.

Exposures during in utero development have the potentialto affect critical developmental steps that may have dramaticeffects not only in the offspring, but in subsequent generations.Two different approaches have been used to study the effectsof obesogens within early developmental windows: exposure offemales throughout pregnancy (from fertilization to birth) (29,37, 61) and exposure at discrete embryonic stages (E8–E14) (62–64, 67). The objective of the latter is to expose the embryos only

FIGURE 1 | Schematic summary of the results from (29). Region 1 represents that genomic areas inaccessible in sperm samples of F3 and F4 mice (A) are

hypomethylated in fat tissue of F4 males (B), and the genes contained in those regions tend to be overexpressed (C). Opposite trends are found in genomic areas

represented by Region 2, with high accessibility in F3 and F4 sperm samples, and hypermethylation and underexpression in fat tissue. Genomic areas depicted by

Region 1 have content of GCs, whereas genomic areas depicted by Region 2 have low GC content (D).

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during the time the primordial germ cells (PGCs) are travelingto the genital ridge, and during which DNA methylation islargely erased (70). However, the benefit of studying the formerapproach is that it allows the exposure to obesogens throughoutthe different phases of embryonic development, all of which maybe important for obesity in later generations.

Another important factor that must be considered whenassessing the effect of environmental pollutants using animalmodels is the concentration the animals are receiving. In orderto be able to extrapolate the information obtained from animalstudies to humans, it is necessary to work with concentrationsof EDCs that are in the realm of what human exposure ismeasured or estimated to be. Typically, the chemical doses atwhich the endocrine system is altered are significantly lower thanthe concentrations that induce toxic effects in the body (44).

SUMMARY AND FUTURE DIRECTIONS

Since the obesogen hypothesis was first proposed, studies inmany laboratories have supported the existence and effectsof obesogens (78). The list of bona fide obesogens (thoseshown to influence obesity, in vivo) continues to grow andmechanisms through which these chemicals act to promoteobesity are emerging. Experimental approaches to study thesemechanisms are improving. In vitro studies continue to providea strong foundation to evaluate the effects of obesogens incells, particularly as more investigators adopt stem cell models,over the limited studies possible in 3T3-L1 preadipocytes. Thesecell culture-based studies offer the possibility to increase thenumbers of chemicals screened and may reveal new mechanismsor hypotheses about the effects of these chemicals using in vivoanimal models. Current technology based on deep sequencingin bulk or in single cells will allow extensive whole-genome

analyses that can provide information critical to understandingthe epigenomic and transcriptomal state of the cells in differenttissues and life stages. A major challenge in this area will beto integrate and interpret these large, multi-omic data sets toprovide the most useful information.

Another major obstacle in understanding the effects ofEDCs and obesogens in humans is the paucity of exposuredata, particularly data from longitudinal prospective cohortstudies. In addition, humans are exposed to mixtures ofEDCs throughout the life course. Fortunately, experimentalresearchers and epidemiologists are teaming up and the adventof “exposome” studies that assess and analyze levels of exposureto obesogens together with a multitude of other chemicals,including EDCs, will enable strong mechanistic links to bemade between chemical exposure and human disease (79). Thiswill inform and guide future laboratory and epidemiologicalapproaches that will overcome the current limitations. In turn,this will allow us to assess the costs to society of EDC andobesogen exposure more accurately. In an optimistic view, thisinformation might influence policy makers to take appropriatesteps to protect the public health.

AUTHOR CONTRIBUTIONS

RC-G and BB participated in the discussion of and wrote theideas reflected in this review.

ACKNOWLEDGMENTS

This work was supported by awards from the National Institutesof Health (ES023316, ES021832) to BB. BB is a named inventoron patents related to nuclear receptors some of which have beenlicensed to for profit entities.

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Conflict of Interest Statement: The authors declare that the research was

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be construed as a potential conflict of interest.

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